Aluminum nanocrystal delivery system, and self-assembled particle adjuvant vaccine based on binding of aluminum nanocrystal delivery system and vaccine antigen molecule

Abstract

The present invention relates to the technical field of biomedicine technology and vaccines, and particularly relates to an aluminum nanocrystal delivery system with a surface covered with an Fc affinity protein and a preparation method for a self-assembled particle adjuvant vaccine. An aluminum nanocrystal is used as a carrier, the surface of the aluminum nanocrystal is covered with an Fc affinity protein molecular layer and an antigen molecule, and the antigen of a recombinant Fc Tag specifically binds to an Fc affinity protein, so that antigen self-assembly is realized, a virus-like particle vaccine is formed, and the antigen density is improved. The present vaccine can generate a high-titer specific antibody by inducing a body fluid and cell immunity.

1 . An aluminum nanocrystal delivery system, wherein the aluminum nanocrystal delivery system is constructed by effectively binding an aluminum nanocrystal to an Fc affinity protein through a modified molecule. 
     
     
         2 . The aluminum nanocrystal delivery system according to  claim 1 , wherein the average particle size of the aluminum nanocrystal is 5-500 nm; and the modified molecule that can be linked to a group of the Fc affinity protein through host-guest coordination and chemoselectivity covalent modification is carried on the surface of the aluminum nanocrystal. 
     
     
         3 . The aluminum nanocrystal delivery system according to  claim 2 , wherein the aluminum nanocrystal is prepared by the following steps:
 fully mixing an aluminum salt solution and a modified molecule solution to obtain a mixed solution; and   adding an alkaline solution to the mixed solution to obtain a reaction solution, adjusting the reaction solution to reach preset pH, then reacting, standing, centrifuging and taking precipitate to obtain the aluminum nanocrystal.   
     
     
         4 . The aluminum nanocrystal delivery system according to  claim 3 , wherein a solute of the aluminum salt solution is more than one of aluminum chloride, aluminum nitrate, aluminum sulfate, and aluminum acetate; the solvent used in the aluminum salt solution is pure water, or a sodium acetate solution with the concentration of 0.01 mol/L; the concentration of the aluminum salt solution is 0.01-0.5 mol/L; and the stirring speed is 100-1400 rpm, and the stirring time is 10 min to 5 h;
 the modified molecule is more than one of L-O-phosphoserine, citric acid, oxalic acid, salicylic acid, and ATP; the concentration of the modified molecule solution is 0.01-0.5 mol/L;   and in the mixed solution, the molar ratio of the aluminum salt to the modified molecule is (10:1)-(1:10);   the alkaline solution is more than one of NaOH, KOH and ammonia solution; the concentration of the alkaline solution is 0.01-0.5 mol/L; the rate of adding the alkaline solution to the mixed solution is 1-20 mL/min; the pH is 5.0-8.0; the reaction temperature is 25-90° C.; the reaction time is 10 min to 10 h; and the standing time is 1-12 h.   
     
     
         5 . The aluminum nanocrystal delivery system according to  claim 1 , wherein the modified molecule comprises a modified molecule that can perform amino, carboxyl and sulfydryl functional group functionalization and chemoselectivity covalent modification group functionalization on a surface functional group of the aluminum nanocrystal. 
     
     
         6 . The aluminum nanocrystal delivery system according to  claim 5 , wherein the modified molecule is selected from more than one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, maleimide, succinimide, azide and alkyne. 
     
     
         7 . A preparation method of an aluminum nanocrystal delivery system according to  claim 1 , comprising the following steps:
 (1) adding an aluminum nanocrystal and a modified molecule to a solvent, and stirring the mixture to allow reaction, thereby obtaining a mixed solution 1;   (2) adding an Fc affinity protein and a modified molecule to a solvent, and stirring the mixture to allow reaction, thereby obtaining a mixed solution 2; and   (3) uniformly mixing the mixed solution 1 in step (1) and the mixed solution 2 in step (2), reacting, centrifugally washing, taking the precipitate, and resuspending to obtain the aluminum nanocrystal delivery system.   
     
     
         8 . The preparation method of an aluminum nanocrystal delivery system according to  claim 7 , wherein the solvent is selected from injection water, ultrapure water, a Tris buffer solution, or a phosphate buffer solution and normal saline; the mass-to-volume ratio of the aluminum nanocrystal to the solvent is 0.5-60 mg/mL; the molar ratio of aluminum ions to the modified molecules in the aluminum nanocrystal is 1:(0.001-1000);
 in step (1), the stirring reaction temperature is 4-25° C., the stirring reaction time is 0.5-24 h, and the stirring rate of the stirring reaction is 100-600 rpm;   in step (2), the stirring reaction temperature is 4-25° C., and the stirring reaction time is 0.5-24 h; and   in step (3), the reaction time is 0.5-24 h, the centrifugal washing is performed twice, and the product is resuspended into the buffer solution with the pH of 7.4-9.   
     
     
         9 . An self-assembled particle adjuvant vaccine based on binding of an aluminum nanocrystal delivery system and a vaccine antigen molecule, wherein the aluminum nanocrystal delivery system is the aluminum nanocrystal delivery system according to  claim 1 , and/or the aluminum nanocrystal delivery system is obtained by the preparation method according to  claim 7 ; and the surface of the aluminum nanocrystal is covered with the Fc affinity protein and the specifically bound self-assembled vaccine antigen molecule. 
     
     
         10 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 9 , wherein the Fc affinity protein is a G protein and/or A protein with high affinity to an Fc region; and the vaccine antigen molecule comprises one or more of an Fc fragment containing pathogenic subunit antigen, a recombinant subunit antigen, an antigen epitope peptide and a nucleic acid antigen. 
     
     
         11 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 10 , wherein the G protein and/or A protein is a bacterial protein or a recombinant protein. 
     
     
         12 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 11 , wherein the bacterial protein is a bacterial protein from Group G  Streptococcus  and/or  Staphylococcus aureus.    
     
     
         13 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 11 , wherein the recombinant protein is a recombinant A protein or G protein, an A protein or G protein with C-terminal recombinant cysteine or phosphoserine, and a polypeptide fragment derived from the A protein and/or G protein. 
     
     
         14 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 10 , wherein the pathogen comprises viruses, bacteria and/or parasites. 
     
     
         15 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 14 , wherein the viruses are selected from DNA virus and/or RNA virus. 
     
     
         16 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 15 , wherein the viruses are selected from coronaviridae, herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, picornaviridae, hepadnaviridae, flaviviridae, papillomaviridae, poxviridae and retroviridae. 
     
     
         17 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 16 , wherein the viruses are selected from novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV. 
     
     
         18 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 14 , wherein the bacteria are selected from gram-positive bacteria and/or gram-negative bacteria. 
     
     
         19 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 18 , wherein the bacteria are selected from  Streptococcus pneumoniae, Haemophilus influenzae, salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter frazier, Pesudomonas pyocyaneum, Acinetobacter baumannii, Mycobacterium tuberculosis  and  Helicobacter pylori.    
     
     
         20 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 19 , wherein the bacteria are selected from  Streptococcus pneumoniae, salmonella, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Helicobacter pylori  and the like. 
     
     
         21 . The self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 14 , wherein the parasites are selected from one or more of plasmodium,  Toxoplasma gondii , trypanosome, schistosome, filariasis and leishmania. 
     
     
         22 . A preparation method of the self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule according to  claim 9 , comprising uniformly mixing a Fc fragment-containing vaccine antigen molecule with an aluminum nanocrystal delivery system solution, so as to obtain a self-assembled particle vaccine based on an aluminum nanocrystal with a surface covered with an Fc affinity protein and an antigen.